Micropatterning of biomolecules, i.e., the attachment of biomolecules within designated regions on solid surfaces while preventing non-specific adhesion at other regions, forms the basis of microarray techniques. Microarray techniques have found many applications in various fields such as diagnostics of diseases (V Devauchelle and G Chiocchia, 2004; C C Xiang and Y D Chen, 2000), drug discovery (S V Chittur, 2004), environmental testing (Y Dharmadi and R Gonzalez, 2004), biological studies (M J Heller, 2002; J C Mills et al, 2001; B Schweitzer and S F Kingsmore, 2002), among others. Tremendous effort has been made by many research groups to develop techniques that are compatible for patterning biomolecules on planar surfaces on the micrometer scale. The most widely used micropatterning methods are pin spotting (P E Sheehan et al, 2003), photoresist lithography (K J Lee et al, 2004) and soft lithography (G M Whitesides, 2001; R S Kane et al, 1999).
Currently most microarrays are fabricated on planar substrates which all involve multi-step surface modifications directly on the substrate. The surface modification is usually achieved by dropping a solution onto the surface or immersing the surface into a solution, and many methods often used to homogenise the solution of reactants and to improve the chemical reactions, such as stirring and vortexing, cannot be carried out on the microarray substrate. For this reason, efficiency of chemical reactions is usually low, resulting in low signal-to-noise (S/N) ratio and poor repeatability of results (T K Jenssen et al, 2003; C Cheng et al, 2003; P Pavluckova et al, 2004).
The use of bead-based materials could be a viable alternative as they are ideal reagent-delivery vehicles providing large reactive surface areas, and versatile methods are available for surface modification of the microbeads. They have become omnipresent in biomedical applications such as immunoassays (T B Martins et al, 2004). Capture reagents are immobilized on the surface of various encoded microbeads, and flow cytometry is applied to detect targets captured by the beads in solution (J D Taylor et al, 2001). Recently, several methods have been developed to produce ordered arrays of beads on a substrate with patterned surfaces to form a random array (R Shen et al, 2005; K Kuhn et al, 2004; E N Warren et al, 2004; H Noda et al, 2003). The capture-reagent-immobilised microbeads are also settled down into etched microwells on optical fiber bundles to form a bead microarray, and a fluorescence signal from each bead (spot) is captured and analysed (A F Jane et al, 2000; S Ferenc et al, 2001). The substrate is used as a template to direct the patterning of the beads. The template may have a patterned surface fabricated using a variety of microfabrication techniques such as optical lithography (M Campbell et al, 2000; V Berger et al, 1997; J A Rogers, 1997), electron (e)-beam lithography (A Last et al, 2004; I Kostic et al, 2003) or imprinting (D Y Khang and H H Lee, 1999; D J Resnick et al, 2003; J D Hoff et al, 2004). However, these microfabrication techniques normally require high-end, very expensive equipment such as mask aligners, e-beam writers, and high-quality clean rooms, as well as well-trained people with specific technical knowledge and experience. Therefore, there is a need in the state of the art for an improved method of fabricating substrates with a patterned surface for patterning. In particular, there is a need for a low-cost non-lithographic method to fabricate substrates with patterned surfaces.
Some methods have been developed for synthesis of porous films. For example, a film with arrays of pores based on self-assembly techniques have been used (O J Cayre and V N Paunov, 2004; J C Jansen et al, 1994), which all include the following steps: self-assembly of colloidal microspheres on a solid substrate to form a 2D crystalline structure as a template (e.g., by filtration, centrifugation, and sedimentation), infusion of other materials into the voids of the self-assembled microspheres, solidification of the material, and removal of the template microspheres through either calcination or solvent extraction, thus creating a 2D solid material with ordered pore arrays. However, the problems associated with these methods are that it is difficult to self-assemble template microspheres with long-range order. Further, any defects will result in a disordered structure of the film. Furthermore, the self-assembled microspheres are not stable and can be easily damaged during the process of removing the template.
Another templating method has been developed based on evaporative cooling and subsequent water-droplet templating to form ordered arrays of “breath figures” and used to produce porous films made of various materials (A Boker et al, 2004; B Francois et al, 1995; J Li et al, 2005; J Peng et al, 2004; O Pitois and B Francois, 1999; G Widawski et al, 1994; M Srinivasarao et al, 2001). Some effort has been made to prepare porous polymer films using the breath-figure method. Francois et al., 1995, prepared polymer films with ordered hexagonal arrays of pores using polystyrene-polyparaphenylene block copolymers, star-like homopolystyrenes, and linear polystyrenes with polar terminal groups in carbon disulphide under a flow of moist gas (G Widawski et al, 1994). Srinivasarao et al, 2001, further developed the method and prepared 2D and 3D materials with multilayers of hexagonally packed pores through a templating mechanism based on thermocapillary convention. The formation of ordered hexagonal arrays of water droplets in polymer films was imaged by Shimomura and co-workers (O Karthaus et al, 2000) and different polymers were used including organic-inorganic hybrid materials (O Karthaus et al, 1999), amphiphilic copolymers (T Nishikawa et al, 1999), organometallics and saccharide-containing polymers (N. Maruyama et al, 1998). The porous films are synthesised in a chamber with well-controlled humidity and with a gas flow over the solution surface. The problem with this method is that conditions such as humidity and gas flow must be properly controlled in order to obtain a porous film with ordered arrays of pores.
There is therefore a need in the state of the art to develop a low-cost non-lithographic method to fabricate substrates with patterned surfaces for patterning microbeads, as well as a method for patterning microbeads.